Fluorescent proteins

ABSTRACT

The present invention relates to novel variants of the fluorescent protein GFP having improved fluorescence properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/619,310 filed on Jul. 19, 2000, which is a continuation of U.S.application Ser. No. 08/819,612, filed on Mar. 17, 1997, now U.S. Pat.No. 6,172,188 B1, which is a continuation of PCT/DK96/00051 filed Jan.31, 1996 and claims priority of Danish Application Ser. No. 1065/95filed Sep. 22, 1995, the contents of which are fully incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to novel variants of the fluorescentprotein GFP having improved fluorescence properties.

BACKGROUND OF THE INVENTION

The discovery that Green Fluorescent Protein (GFP) from the jellyfish A.victoria retains its fluorescent properties when expressed inheterologous cells has provided biological research with a new, uniqueand powerful tool (Chalfie et al (1994). Science 263:802; Prasher (1995)Trends in Genetics 11:320; WO 95/07463).

Furthermore, the discovery of a blue fluorescent variant of GFP (Heim etal. (1994). Proc.Natl.Acad.Sci. 91:12501) has greatly increased thepotential applications of using fluorescent recombinant probes tomonitor cellular events or functions, since the availability of probeshaving different excitation and emission spectra permits simultaneousmonitoring of more than one process.

However, the blue fluorescing variant described by Heim et al, Y66H-GFP,suffers from certain limitations: The blue fluorescence is weak(emission maximum at 448 nm), thus making detection difficult, andnecessitating prolonged excitation of cells expressing Y66H-GFP.Moreover, the prolonged period of excitation is damaging to cellsespecially because the excitation wavelength is in the UV range, 360nm-390 nm.

A very important aspect of using recombinant, fluorescent proteins instudying cellular functions is the non-invasive nature of the assay.This allows detection of cellular events in intact, living cells. Alimitation with current fluorescent proteins is, however, thatrelatively high intensity light sources are needed for visualization.Especially with the blue variant, Y66H-GFP, it is necessary to excitewith intensities that are damaging to most cells. It is worth mentioningthat some cellular events like oscillations in intracellular signallingsystems, e.g. cytosolic free calcium, are very photo sensitive. Afurther consequence of the low light emittance is that only high levelsof expression can be detected. Obtaining such high level expression maystress the transcriptional and/or translational machinery of the cells.

The excitation spectrum of the green fluorescent protein from AequoreaVictoria shows two peaks: A major peak at 396 nm, which is in thepotentially cell damaging UV range, and a lesser peak at 475 nm, whichis in an excitation range that is much less harmful to cells. Heim etal.(1995), Nature, Vol. 373, p. 663-4, discloses a Ser65Thr mutation ofGFP (S65T) having longer wavelengths of excitation and emission, 490 nmand 510 nm, respectively, than the wild-type GFP and wherein thefluorophore formation proceeded about fourfold more rapidly than in thewild-type GFP.

Expression of GFP or its fluorescent variants in living cells provides avaluable tool for studying cellular events and it is well known thatmany cells, including mammalian cells, are incubated at approximately37° C. in order to secure optimal and/or physiologically relevantgrowth. Cell lines originating from different organisms or tissues mayhave different relevant temperatures ranging from about 35° C. forfibroblasts to about 38° C.-39° C. for mouse β-cells. Experience hasshown, however, that the fluorescent signal from cells expressing GFP isweak or absent when said cells are incubated at temperatures above roomtemperature, cf. Webb, C. D. et al., Journal of Bacteriology, October1995, p. 5906-5911. Ogawa H. et al., Proc. Natl. Acad. Sci. USA, Vol.92, pp. 11899-11903, December 1995, and Lim et al. J. Biochem. 118,13-17 (1995). The improved fluorescent variant S65T described by Heim etal. (1995) supra also displays very low fluorescence when incubatedunder normal culture conditions (37° C.), cf. Kaether and Gerdes FEBSLetters 369 (1995) pp. 267-271. Many experiments involving the study ofcell metabolism are dependent on the possibility of incubating the cellsat physiologically relevant temperatures, i.e. temperatures at about 37°C.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide novel fluorescentproteins, such as F64L-GFP (SEQ ID NO: 18, hereinafter referred to asF64L-GFP), F64L-Y66H-GPP (SEQ ID NO: 16, hereinafter referred to asF64L-Y66H-GFP) and F64L-S65T-GFP (SEQ ED NO: 20, hereinafter referred toas F64L-S65T-GFP) that result in a cellular fluorescence far exceedingthe cellular fluorescence from cells expressing the parent proteins,i.e. GFP (SEQ ID NO: 22, hereinafter referred to as GFP), the bluevariant Y66H-GFP and the S65T-GFP variant, respectively. This greatlyimproves the usefulness of fluorescent proteins in studying cellularfunctions in living cells.

A further purpose of the invention is to provide novel fluorescentproteins that exhibit high fluorescence in cells expressing them whensaid cells are incubated at a temperature of 30° C. or above, preferablyat a temperature of from 32° C. to 39° C., more preferably at atemperature of from 35° C. to 38° C., and most preferably at atemperature of about 37° C.

It is known that fluorescence in wild-type GFP is due to the presence ofa chromophore, which is generated by cyclisation and oxidation of theSYG at position 65-67 in the predicted primary amino acid sequence andpresumably by the same reasoning of the SHG sequence and other GFPanalogues at positions 65-67, cf. Heim et al. (1994). Surprisingly, wehave found that a mutation, preferably a substitution, of the F aminoacid residue at position 1 preceding the S of the SYG or SHG chromophoreor the T of the THG chromophore, in casu position 64 in the predictedprimary amino acid sequence, results in a substantial increase offluorescence intensity apparently without shifting the excitation andemission wavelengths. This increase is remarkable for the blue variantY66H-GFP, which hitherto has not been useful in biological systemsbecause of its weak fluorescence.

The F64L, F64I, F64V, F64A, and F64G substitutions are preferred, theF64L substitution being most preferred, but other mutations, e.g.deletions, insertions, or posttranslational modifications immediatelypreceding the chromophore are also included in the invention, providedthat they result in improved fluorescence properties of the variousfluorescent proteins. It should be noted that extensive deletions mayresult in loss of the fluorescent properties of GFP. It has been shown,that only one residue can be sacrificed from the amino terminus and lessthan 10 or 15 from the carboxyl terminus before fluorescence is lost,cf. Cubitt et al. TIBS Vol. 20 (11), pp. 448-456, November 1995.

Accordingly, one aspect of the present invention relates to afluorescent protein derived from Aequorea Green Fluorescent Protein(GFP) or any functional analogue thereof, wherein the amino acid inposition 1 upstream from the chromophore has been mutated to provide anincrease of fluorescence intensity when the fluorescent protein of theinvention is expressed in cells. Surprisingly, said mutation alsoresults in a significant increase of the intensity of the fluorescentsignal from cells expressing the mutated GFP and incubated at 30° C. orabove 30° C., preferably at about 37° C., compared to the prior art GFPvariants.

There are several advantages of the proteins of the invention,including:

Excitation with low energy light sources. Due to the high degree ofbrightness of F64L-Y66H-GFP and F64L-GFP their emitted light can bedetected even after excitation with low energy light sources. Thereby itis possible to study cellular phenomena, such as oscillations inintracellular signalling systems, that are sensitive to light induceddamage. As the intensity of the emitted light from the novel blue andgreen emitting fluorescent proteins are of the same magnitude, it ispossible to visualize them simultaneously using the same light source.

A real time reporter for gene expression in living cells is nowpossible, since the fluorescence from F64L-Y66H-GFP and F64L-GFP reachesa detectable level much faster than from wild type GFP, and prior knownderivatives thereof. Hence, it is more suitable for real time studies ofgene expression in living cells. Detectable fluorescence may be obtainedfaster due to shorter maturation time of the chromophore, higheremission intensity, or a more stable protein or a combination thereof.

Simultaneous expression of the novel fluorescent proteins under controlof two or more separate promoters.

Expression of more than one gene can be monitored simultaneously withoutany damage to living cells.

Simultaneous expression of the novel proteins using one reporter asinternal reference and the other as variable marker, since regulatedexpression of a gene can be monitored quantitatively by fusion of apromoter to e.g. F64L-GFP (or F64L-Y66H-GFP), measuring thefluorescence, and normalizing it to the fluorescence of constitutivelyexpressed F64L-Y66H-GFP (or F64L-GFP). The constitutively expressedF64L-Y66H-GFP (or F64L-GFP) works as internal reference.

Use as a protein tag in living and fixed cells. Due to the strongfluorescence the novel proteins are suitable tags for proteins presentat low concentrations. Since no substrate is needed and visualisation ofthe cells do not damage the cells dynamic analysis can be performed.

Use as an organelle tag. More than one organelle can be tagged andvisualised simultaneously in living cells, e.g. the endoplasmicreticulum and the cytoskeleton.

Use as markers in cell or organelle fusions. By labelling two or morecells or organelles with the novel proteins, e.g. F64L-Y66H-GFP andF64L-GFP, respectively, fusions, such as heterokaryon formation, can bemonitored.

Translocation of proteins fused to the novel proteins of the inventioncan be visualised. The translocation of intracellular proteins to aspecific organelle, can be visualised by fusing the protein of interestto one fluorescent protein, e.g. F64L-Y66H-GFP, and labelling theorganelle with another fluorescent protein, e.g. F64L-GFP, which emitslight of a different wavelength. Translocation can then be detected as aspectral shift of the fluorescent proteins in the specific organelle.

Use as a secretion marker. By fusion of the novel proteins to a signalpeptide or a peptide to be secreted, secretion may be followed on-linein living cells. A precondition for that is that the maturation of adetectable number of novel fluorescent protein molecules occurs fasterthan the secretion. This appears not to be the case for the fluorescentproteins GFP or Y66H-GFP of the prior art.

Use as genetic reporter or protein tag in transgenic animals. Due to thestrong fluorescence of the novel proteins, they are suitable as tags forproteins and gene expression, since the signal to noise ratio issignificantly improved over the prior art proteins, such as wild-typeGFP.

Use as a cell or organelle integrity marker. By co-expressing two of thenovel proteins, the one targeted to an organelle and the other expressedin the cytosol, it is possible to calculate the relative leakage of thecytosolic protein and use that as a measure of cell integrety.

Use as a marker for changes in cell morphology. Expression of the novelproteins in cells allows easy detection of changes in cell morphology,e.g. blebbing, caused by cytotoxic agents or apoptosis. Suchmorphological changes are difficult to visualize in intact cells withoutthe use of fluorescent probes.

Use as a transfection marker, and as a marker to be used in combinationwith FACS sorting. Due to the increased brightness of the novel proteinsthe quality of cell detection and sorting can be significantly improved.

Use of the novel proteins as a ratio real-time kinase probe. Bysimultaneous expression of, e.g. F64L-GFP (or F64L-Y66H-GFP), whichemits more light upon phophorylation and a derivative of F64L-Y66H-GFPwhich emits less light upon phophorylation. Thereby, the ratio of thetwo intensities would reveal kinase activity more accurately than onlyone probe.

Use as real-time probe working at near physiological concentrations.Since the novel proteins are significantly brighter than wild type GFPand prior art derivatives at about 37° C. the concentration needed forvisualisation can be lowered. Target sites for enzymes engineered intothe novel proteins, e.g. F64L-Y66H-GFP or F64L-GFP, can therefore bepresent in the cell at low concentrations in living cells. This isimportant for two reasons: 1) The probe must interfere as little aspossible with the intracellular process being studied; 2) thetranslational and transcriptional apparatus should be stressedminimally.

The novel proteins can be used as real time probes based on energytransfer. A probe system based on energy transfer from, e.g.F64L-Y66H-GFP to F64L-GFP.

The novel proteins can be used as reporters to monitor live/dead biomassof organisms, such as fungi. By constitutive expression of F64L-Y66H-GFPor F64L-GFP in fungi the viable biomass will light up.

Transposon vector mutagenesis can be performed using the novel proteinsas markers in transcriptional and translational fusions.

Transposons to be used in microorganisms encoding the novel proteins.The transposons may be constructed for translational and transcriptionalfusions. To be used for screening for promoters.

Transposon vectors-encoding the novel proteins, such as F64L-Y66H-GFPand F64L-GFP, can be used for tagging plasmids and chromosomes.

Use of the novel proteins enables the study of transfer of conjugativeplasmids, since more than one parameter can be followed in living cells.The plasmid may be tagged by F64L-Y66H-GFP or F64L-GFP and thechromosome of the donor/recipient by F64L-Y66H-GFP or F64L-GFP.

Use as a reporter for bacterial detection by introducing the novelproteins into the genome of bacteriophages.

By engineering the novel proteins, e.g. F64L-Y66H-GFP or F64L-GFP, intothe genome of a phage a diagnostic tool can be designed. F64L-Y66H-GFPor F64L-GFP will be expressed only upon transfection of the genome intoa living host. The host specificity is defined by the bacteriophage.

Any novel feature or combination of features described herein isconsidered essential to this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows a map of pUC 19-GFP plasmid construction;

FIG. 2a is the DNA (SEQ ID NO: 21) and predicted primary amino acidsequence (SEQ ID NO: 22) of GFP;

FIG. 2b is the nucleotide sequence of GFP (SEQ ID NO: 21);

FIG. 3 is the DNA (SEQ ID NO: 15) and predicted amino acid sequence (SEQID NO: 16) and predicted amino acid sequence of (SEQ ID NO: 16) ofF64L-Y66H-GFP;

FIG. 4 is the DNA (SEQ ID NO: 17) and predicted amino acid sequence (SEQED NO: 18) of F64L-GFP;

FIG. 5 is the DNA (SEQ ID NO: 19) and predicted amino acid sequence (SEQID NO: 20) of F64L-S65T-GFP;

FIG. 6a is a graph of fluorescence emission spectra measured in cellsgrown at 22° C. for 16 hours and excited with light at 398 nm forF64L-GFP, GFP, GFP-N1, F64L-S65T-GFP, and lacZ;

FIG. 6b is a graph of fluorescence emission spectra measured in cellsgrown at 37° C. for 16 hours and excited with light at 398 nm forF64L-GFP, GFP, GFP-N1, F64L-S65T-GFP, and lacZ;

FIG. 6c is a graph of fluorescence emission spectra measured in cellsgrown at 22° C. for 16 hours and excited with light at 470 nm forF64L-GFP, GFP, GFP-N1, F64L-S65T-GFP, and lacZ;

FIG. 6d is a graph of fluorescence emission spectra measured in cellsgrown at 37° C. for 16 hours and excited with light at 470 nm forF64L-GFP, GFP, GFP-N 1, F64L-S65T-GFP, and lacZ;

FIG. 6e is a graph of fluorescence emission spectra measured in cellsgrown at 22° C. for 16 hours and excited with light at 380 nm forF64L-Y66H-GFP, Y66H-GFP and lacZ;

FIG. 6f is a graph of fluorescence emission spectra measured in cellsgrown at 37° C. for 16 hours and excited with light at 380 nm forF64L-Y66H-GFP, Y66H-GFP and lacZ.

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment of the present invention, the novelfluorescent protein is the F64L mutant of GFP or the blue variantY66H-GFP, said mutant showing increased fluorescence intensity. Apreferred sequence of the gene encoding GFP derived from Aequoreavictoria is disclosed in FIG. 2 herein. FIG. 2 shows the nucleotidesequence of a wild-type GFP (Hind3-EcoR1 fragment) and the amino acidsequence, wherein start codon ATG corresponds to position 8 and stopcodon TAA corresponds to position 722 in the nucleotide sequence. Amicroorganism, E. coli NN049087, carrying the DNA sequence shown in FIG.2 has been deposited for the purpose of patent procedure according tothe Budapest Treaty in Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Mascheroderweg 1 b, D-38124 Braunschweig, FederalRepublic of Germany, under the deposition No. DSM 10260. Anothersequence of an isotype of this gene is disclosed by Prasher et al., Gene111, 1992, pp. 229-233 (GenBank Accession No. M62653). Besides, thenovel fluorescent proteins may also be derived from other fluorescentproteins, e.g. the fluorescent protein of the sea pansy Renillarenziformis.

Herein the abbreviations used for the amino. acids are those stated inJ. Biol. Chem. 243 (1968), 3558.

The DNA construct of the invention encoding the novel fluorescentproteins may be prepared synthetically by established standard methods,e.g. the phosphoamidite method described by Beaucage and Caruthers,Tetrahedron Letters 22 (1981), 1859-1869, or the method described byMatthes et al., EMBO Journal 3 (1984), 801-805. According to thephosphoamidite method, oligonucleotides are synthesized, e.g. in anautomatic DNA synthesizer, purified, annealed, ligated and cloned insuitable vectors.

The DNA construct may also be prepared by polymerase chain reaction(PCR) using specific primers, for instance as described in U.S. Pat. No.4,683,202 or Saiki et al., Science 239 (1988), 487-491. A more recentreview of PCR methods may be found in PCR Protocols, 1990, AcademicPress, San Diego, Calif., USA.

The DNA construct of the invention may be inserted into a recombinantvector which may be any vector which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the fluorescent protein of the invention is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid of viral DNA, or maycontain elements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the fluorescent protein ofthe invention.

The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or hererologous to the host cell,including native Aequorea GFP genes.

Examples of suitable promoters for directing the transcription of theDNA sequence encoding the fluorescent protein of the invention inmammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol.1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiteret al., Science 222 (1983), 809-814) or the adenovirus 2 major latepromoter.

An example of a suitable promoter for use insect cells is the polyhedrinpromoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311,(1992) 7-11), the P10 promoter (J.M. Vlak et al. J. Gen. Virology 69,1988, pp. 765-776), the Autographa califormica polyhedrosis virus basicprotein promoter (EP 397 485), the baculovirus immediate early gene 1promoter (U.S. Pat. Nos. 5,155,037; 5,162,222), or the baculovirus 39Kdelayed-early gene promoter (U.S. Pat. Nos. 5,155,037; 5,162,222).

Examples of suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No.4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654)promoters.

Examples of suitable promoters for use in filamentous fungus host cellsare, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4(1985), 2093-2099) or the tpiA promoter. Examples of other usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters.

Examples of suitable promoters for use in bacterial host cells includethe promoter of the Bacillus stearorhennophilus maltogenic amylase gene,the Bacillus lichenziformis alpha-amylase gene, the Bacillusamyloliquefaciens BAN amylase gene, the Bacillus subtilis alkalineprotease gene, or the Bacillus pumilus xylosidase gene, or by the phageLambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters.

The DNA sequence encoding the novel fluorescent proteins of theinvention may also, if necessary, be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et op. cit.) terminators. The vector may furthercomprise elements such as polyadenylation signals (e.g. from SV40 or theadenovirus 5 Elb region), transcriptional enhancer sequences (e.g. theSV40 enhancer) and translational enhancer sequences (e.g. the onesencoding adenovirus VA RNAs).

The recombinant vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. An example of such asequence (when the host cell is a mammalian cell) is the SV40 origin ofreplication.

When the host cell is a yeast cell, suitable sequences enabling thevector to replicate are the yeast plasmid 2μ replication genes REP 1-3and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin or hygromycin. For filamentousfungi, selectable markers include amdS, pyrG, argB, niaD, sC.

The procedures used to ligate the DNA sequences coding for thefluorescent protein of the invention, the promoter and optionally theterminator and/or secretory signal sequence, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op.cit.).

The host cell into which the DNA construct or the recombinant vector ofthe invention is introduced may be any cell which is capable ofexpressing the present DNA construct and includes bacteria, yeast, fungiand higher eukaryotic cells.

Examples of bacterial host cells which, on cultivation, are capable ofexpressing the DNA construct of the invention are grampositive bacteria,e.g. strains of Bacillus, such as B. subtilis, B. licheniformis, B.lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatheriumor B. thuringiensis, or strains of Streptomyces, such as S. lividans orS. murinus, or gramnegative bacteria such as Echerichia coli. Thetransformation of the bacteria may be effected by protoplasttransformation or by using competent cells in a manner known per se (cf.Sambrook et al., supra).

Examples of suitable mammalian cell lines are the HEK293 and the HeLacell lines, primary cells, and the COS (e.g. ATCC CRL 1650), BHK (e.g.ATCC CRL 1632, ATCC CCL 10), CHL (e.g. ATCC CCL39) or CHO (e.g. ATCC CCL61) cell lines. Methods of transfecting mammalian cells and expressingDNA sequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol.Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci.USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro andPearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb,Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Examples of suitable yeast cells include cells of Saccharomyces spp. orSchizosaccharomyces spp., in particular strains of Saccharomycescerevisiae or Saccharomyces kluyveri. Methods for transforming yeastcells with heterologous DNA and producing heterologous polypeptidestherefrom are described, e.g. in U.S. Pat. No. 4,599,311, No. 4,931;373,No. 4,870,008, No. 5,037,743, and No. 4,845,075, all of which are herebyincorporated by reference. Transformed cells are selected by a phenotypedetermined by a selectable marker, commonly drug resistance or theability to grow in the absence of a particular nutrient, e.g. leucine. Apreferred vector for use in yeast is the POT1 vector disclosed in U.S.Pat. No. 4,931,373. The DNA sequence encoding the fluorescent protein ofthe invention may be preceded by a signal sequence and optionally aleader sequence , e.g. as described above. Further examples of suitableyeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula,e.g. H. polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J.Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).

Examples of other fungal cells are cells of filamentous fungi, e.g.Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., inparticular strains of A. oryzae, A. nidulans or A. niger. The use ofAspergzllus spp. for the expression of proteins is described in, e.g.,EP 272 277, EP 230 023, EP 184 438.

When a filamentous fungus is used as the host cell, it may betransformed with the DNA construct of the invention, conveniently byintegrating the DNA construct in the host chromosome to obtain arecombinant host cell. This integration is generally considered to be anadvantage as the DNA sequence is more likely to be stably maintained inthe cell. Integration of the DNA constructs into the host chromosome maybe performed according to conventional methods, e.g. by homologous orheterologous recombination.

Transformation of insect cells and production of heterologouspolypeptides therein may be performed as described in U.S. Pat. No.4,745,051; No. 4,879,236; No. 5,155,037; 5,162,222; EP 397,485) all ofwhich are incorporated herein by reference. The insect cell line used asthe host may suitably be a Lepidoptera cell line, such as Spodopterafrugiperda cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214).Culture conditions may suitably be as described in, for instance, WO89101029 or WO 89/01028, or any of the aforementioned references.

The transformed or transfected host cell described above is thencultured in a suitable nutrient medium under conditions permitting theexpression of the present DNA construct after which the cells may beused in the screening method of the invention. Alternatively, the cellsmay be disrupted after which cell extracts and/or supernatants may beanalysed for fluorescence.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection).

In the method of the invention, the fluorescence of cells transformed ortransfected with the DNA construct of the invention may suitably bemeasured in a spectrometer or a fluorescence microscope where thespectral properties of the cells in liquid culture may be determined asscans of light excitation and emission.

The invention is further illustrated in the following examples withreference to the appended drawings.

EXAMPLE 1

Cloning of cDNA Encoding GFP

Briefly, total RNA, isolated from A. victoria by a standard procedure(Sambrook et al., Molecular Cloning. 2., eds. (1989) (Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.), 7.19-7.22) was convertedinto cDNA by using the AMV reverse transcriptase (Promega, Madison,Wis., USA) as recommended by the manufacturer. The cDNA was then PCRamplified, using PCR primers designed on the basis of a previouslypublished GFP sequence (Prasher et al., Gene 111 (1992), 229-233;GenBank accession No. M62653) together with the UlTma™ polymerase(Perkin Elmer, Foster City, Calif., USA). The sequences of the primerswere: GFP-2 (SEQ ID NO: 1): TGGAATAAGCTTTATGAGTAAAGGAGAAGAACTTTT andGFP-1 (SEQ ID NO:2): AAGAATTCGGATCCCTTTAGTGTCAATTGGAAGTCT.

Restriction endonuclease sites inserted in the 5′ (a HindIII site) and3′ (EcoRI and BamHI sites) primers facilitated the cloning of the PCRamplified GEP cDNA into a slightly modified pUC19 vector. The details ofthe construction are as follows: LacZ Shine-Dalgarno AGGA, immediatelyfollowed by the 5′ HindIII site plus an extra T and the GEP ATG codon,giving the following DNA sequence (SEQ ID NO: 23) at the lacZ-promoterGFP fusion point: P_(LaCZ)-AGGAAAGCTTTATG-GFP. At the 3′ end of the GFPcDNA, the base pair corresponding to nucleotide 770 in the published GFPsequence (GenBank accession No. M62653) was fused to the EcoR1 site ofthe pUC19 multiple cloning site (MCS) through a PCR generated BamHI,EcoRI linker region).

The DNA sequence and predicted primary amino acid sequence of GFP isshown below in FIG. 2a. Another DNA sequence encoding the same aminoacid sequence as shown in FIG. 2a is shown in FIG. 2b. To generate theblue fluorescent variant described by Heim et al. (1994), a PCR primerincorporating the Y66H substitution responsible for changing greenfluorescence into blue fluorescence was used as 5′ PCR primer incombination with a GFP specific 3′ primer. The template was the GFPclone described above. The sequence of the 5′ primer is5′-CTACCTGTTCCATGGCCAACGCTTGTCACTACTTTCCTCATGGTGTTCAATGCTTTTCTAGATACCC-3′ (SEQ ID NO:3). Its 5′ end corresponds to position 164 inthe GFP sequence. In addition to the Y66H substitution, the 5′ primerintroduces a A to T change at position 223; this mutation creates a Xba1site without changing an amino acid. The 5′ primer also contains thenaturally occuring Nco1 recognition sequence (position 173 in the GFPsequence). The sequence of the 3′ primer is5′-AAGAATTCGGATCCCTTTAGTGTCAATTGGAAGTCT-3′ (SEQ ID NO:4). Position 3from the 5′ end is the first base of the EcoR1 recognition site thatcorresponds to the 3′ end of the GFP sequence. The resulting PCR productwas digested with Nco1 and EcoR1 and cloned into an Nco1-EcoR1 vectorfragment to reconstitute the entire Y66H-GFP gene.

E. coli cells carrying an expression vector containing Y66H-GFP weregrown overnight in the presence of 10 micrograms per mlN-methyl-N-nitro-N-nitrosoguanidine. Plasmid DNA was isolated, the 764bp Hind3-EcoR1 insert containing Y66H-GFP was isolated and cloned into aHind3-EcoR1 digested vector fragment, allowing expression of the insertin E. coli. E. coli transformants were inspected for blue fluorescencewhen excited with a 365 nm UV light, and colonies that appeared tofluoresce stronger than wildtype BFP were identified.

10 ng DNA from one particular colony was used as template in a PCRreaction containing 1.5 units of Taq polymerase (Perkin Elmer), 0.1 mMMnCl₂, 0.2 mM each of dGTP, dCTP and dTTP, 0.05 mM dATP, 1.7 mM MgCl₂and the buffer recommended by the manufacturer. The primers used flankthe Y66H-GFP insert. The sequence of the 5′ primer was5′-AATTGGTACCAAGGAGGTAAGCTTTATGAG-3′ (SEQ ID NO:5); it contains a Hind3recognition sequence. The sequence of the 3′ primer was5′-CTTTCGTTTTGAATTCGGATCCCTTTAGTG-3′ (SEQ ID NO:6); it contains a EcoR1recognition sequence.

The PCR product was digested with Hind3 and EcoR1 and cloned into aHind3-EcoR1 digested vector fragment, allowing expression of the insertin E.coli.E.coli transformants were inspected for blue fluorescence whenexcited with a 365 nm UV light, and colonies that appeared to fluorescestronger than Y66H-GFP were identified.

Plasmid DNA from one strongly fluorescing colony (called BX12-1A) wasisolated and the Y66H-GFP insert was subjected to sequencedetermination. The mutation F64L was identified. This mutation replacesthe phenylalanine residue preceding the SHG tripeptide chromophoresequence of Y66H-GFP with leucine. No other aminoacid changes werepresent in the Y66H-GFP sequence of BX12-1A. The DNA sequence andpredicted primary amino acid sequence of F64L-Y66H-GFP is shown in FIG.3 below.

EXAMPLE 2

F64L-GFP was constructed as follows: An E.coli expression vectorcontaining Y66H-GFP was digested with restriction enzymes Nco1 and Xba1.The recognition sequence of Nco1 is located at position 173 and therecognition sequence of Xba1 is located at position 221 in theF64L-Y66H-GFP sequence listed below. The large Nco1-Xba1 vector fragmentwas isolated and ligated with a synthetic Nco1-Xba1 DNA linker of thefollowing sequence:

One DNA strand has the sequence:

5′-CATGGCCAACGCTTGTCACTACTCTCTCTTATGGTGTTCAATGCTTTT-3′ (SEQ ID NO:7)

The other DNA strand has the sequence:

5′-CTAGAAAAGCATTGAACACCATAAGAGAGAGTAGTGACAAGCGTTGGC-3′ (SEQ ID NO:8)

Upon annealing, the two strands form a Nco1-Xba1 fragment thatincorporates the sequence of the GFP chromophore SYG with the F64Lsubstitution preceding SYG. The DNA sequence and predicted primary aminoacid sequence of F64L-GFP is shown in FIG. 4 below.

The S65T-GFP mutation was described by Heim et al (Nature vol.373 pp.663-664, 1995). F64L-S65T-GFP was constructed as follows: An E.coliexpression vector containing Y66H-GFP was digested with restrictionenzymes Nco1 and Xba1. The recognition sequence of Nco1 is located atposition 173 and the recognition sequence of Xba1 is located at position221 in the F64L-Y66H-GFP sequence listed below. The large Nco1-Xba1vector fragment was isolated and ligated with a synthetic Nco1-Xba1 DNAlinker of the following sequence:

One DNA strand has the sequence:

5′-CATGGCCAACGCTTGTCACTACTCTCACTTATGGTGTTCAATGCTTTT-3′ (SEQ ID NO:9)

The other DNA strand has the sequence:

5′-CTAGAAAAGCATTGAACACCATAAGTGAGAGTAGTGACAAGCGTTGGC-3′ (SEQ ID NO:10).

Upon annealing, the two strands form a Nco1-Xba1 fragment thatincorporates the F64L and S65T mutations in the GFP chromophore. The DNAsequence and predicted primary amino acid sequence of F64L-S65T-GFP isshown in FIG. 5 below.

The E. coli expression vector contains an IPTG(isopropyl-thio-galactoside)-inducible promoter. The E. coli strain usedis a del(lacZ)MI5 derivative of K 803 (Sambrook et al. supra).

The GFP allele present in the pGFP-N1 plasmid (available from ClontechLaboratories) was introduced into the IPTG inducible E.coli expressionvector in the following manner:

1 ng pGFP-N1 plasmid DNA was used as template in a standard PCR reactionwhere the 5′ PCR primer had the sequence:

5′-TGGAATAAGCTTTATGAGTAAAGGAGAAGAACTTTT-3′ (SEQ ID NO:11)

and the 3′ PCR primer had the sequence:

5′-GAATCGTAGATCTTTATTTGTATAGTTCATCCATG-3′ (SEQ ID NO:12).

The primers flank the GFP-N1 insert in the vector pGFP-N1. The 5′ primerincludes the ATG start codon preceded by a Hind3 cloning site. The 3′primer includes a TAA stop codon followed by a Bgl2 cloning site.

The PCR product was digested with Hind3 and Bgl2 and cloned into aHind3-BamH1digested vector fragment behind an IPTG inducible promoter,allowing expression of the insert in E.coli in the presence of IPTG.

The lacZ gene present in the pZeoSV-LacZ plasmid (available fromInvitrogen) was introduced into the IPTG inducible E.coli expressionvector in the following manner:

1 ng pZeoSV-LacZ plasmid DNA was used as template in a standard PCRreaction where the 5′ PCR primer had the sequence:

5′-TGGAATAAGCTTTATGGATCCCGTCGTTTTACAACGTCGT-3′ (SEQ ID NO:13)

and the 3′ PCR primer had the sequence:

5′-GCGCGAATTCTTATTATTATTTTTGACACCAGAC-3′ (SEQ ID NO:14).

The primers flank the lacZ insert in the vector pZeoSV-LacZ. The 5′primer includes the ATG start codon preceded by a Hind3 cloning site.The 3′ primer includes a TAA stop codon followed by an EcoR1 cloningsite.

The PCR product was digested with Hind3 and EcoR1 and cloned into aHind3-EcoR1 digested vector fragment behind an IPTG inducible promoter,allowing expression of the insert in E.coli in the presence of IPTG.

To measure and compare the fluorescence generated in E. coli cellsexpressing GFP, GFP-N1, F64L-GFP, F64L-S65T-GFP, Y66H-GFP, F64L-Y66H-GFPor beta-galactosidase (as background control) under various conditionsthe following experiments were done:

E. coli cells containing an expression plasmid allowing expression ofone of the various gene products upon induction with IPTG were grown inLB medium containing 100 micrograms per milliliter ampicillin and noIPTG. To 1 ml cell suspension was added 0.5 ml 50% glycerol and cellswere frozen and kept frozen at −80 C.

Cells from the −80 C glycerol stocks were inoculated into 2 ml LB mediumcontaining 100 μg/ml ampicillin and grown with aeration at 37 C for 6hours. 2 microliters of this inoculum was transferred to each of twotubes containing 2 ml of LB medium with 100 μg/ml ampicillin and 1 nMIPTG. The two sets of tubes were incubated with aeration at twodifferent temperatures: room temperature (22 C) and 37 C.

After 16 hours 0.2 ml samples were taken of cells expressing GFP,GFP-N1, F64L-GFP, F64L-S65T-GFP, Y66H-GFP, F64L-Y66H-GFP- orbeta-galactosidase. Cells were pelleted, the supernatant was removed,cells were resuspended in 2 ml water and transferred to a cuvette.Fluorescence emission spectra were measured in a LS-50 luminometer(Perkin-Elmer) with excitation and emission slits set to 10 nm. Theexcitation wavelengths were set to 398 nm and 470 nm for GFP, GFP-N1,F64L-GFP and F64L-S65T-GFP; 398 nm is near the optimal excitationwavelength for GFP, GFP-N1 and F64L-GFP, and 470 nm is near the optimalexcitation wavelength for F64L-S65T-GFP. For Y66H-GFP and F64L-Y66H-GFPthe excitation wavelength was set to 380 nm, which is near the optimalexcitation wavelength for these derivatives. Beta-galactosidaseexpressing cells were included as background controls. Following themeasurements in the LS-50 luminometer, the optical density at 450 nm wasmeasured for each sample in a spectrophotometer (Lambda UV/VIS,Perkin-Elmer). This is a measure of total cells in the assay.Luminometer data were normalized to the optical density of the sample.

The results of the experiments are shown in FIGS. 6a-6 f below and canbe summarized as follows:

After 16 hours at 22 C using an excitation wavelength of 398 nm therewere large signals for GFP and F64L-GFP, and detectable signals forGFP-N1 and F64L-S65T-GFP, cf. FIG. 6a.

After 16 hours at 37 C with an excitation wavelength of 398 nm there wasa large signals for F64L-GFP, a detectable signal for F64L-S65T-GFP, andno detectable signals for GFP and GFP-N1, cf. FIG. 6b.

After 16 hours at 22 C with an excitation wavelength of 470 nm there wasa large signals for F64L-S65T-GFP, detectable signals for GFP andF64L-GFP, and no detectable signals for GFP-N1, cf. FIG. 6c.

After 16 hours at 37 C with an excitation wavelength of 470 nm therewere large signals for F64L-S65T-GFP and F64L-GFP, and no detectablesignals for GFP and GFP-N1, cf. FIG. 6d.

After 16 hours at 22 C with an excitation wavelength of 380 nm therewere detectable signals over background for Y66H-GFP and F64L-Y66H-GFP,cf. FIG. 6e.

After 16 hours at 37 C with an excitation wavelength of 380 nm there wasno detectable signal over background for Y66H-GFP and a large signal forF64L-Y66H-GFP, cf. FIG. 6f.

To determine whether the differences in fluorescence signals were due todifferences in expression levels, total protein from the E.coli cells'(0.5 OD₄₅₀ units) analyzed as described above was fractionated bySDS-polyacrylamide gel electrophoresis (12% Tris-glycine gels fromBIO-RAD Laboratories) followed by Western blot analysis (ECL Westernblotting from Amersham International) with polyclonal GFP antibodies(from rabbit). The result showed that expression levels of GFP, GFP-N1,F64L-GFP, F64L-S65T-GFP, Y66H-GFP and F64L-Y66H-GFP were identical, bothat 22 C and 37 C. The differences in fluorescence signals are thereforenot due to different expression levels.

EXAMPLE 3

Influence of the F64L Substitution on GFP and its Derivatives whenExpressed in Mammalian Cells.

F64L-Y66H-GFP, F64L-GFP, and F64L-S65T-GFP were cloned into pcDNA3(Invitrogen, Calif., USA) so that the expression was under control ofthe CMV promoter. Wild-type GFP was expressed from the pGFP-N1 plasmid(Clontech, Calif., USA) in which the CMV promoter controls theexpression. Plasmid DNA to be used for transfection were purified usingJetstar Plasmid kit (Genomed Inc. N.C., USA) and was dissolved indistilled water.

The precipitate used for the transfections were made by mixing thefollowing components: 2 μg DNA in 44 μl of water were mixed with 50 μl2×HBS buffer (280 mM NaCl, 1.5 mM Na₂HPO₄, 12 mM dextrose, 50 mM HEPES)and 6.2 μl 2M CaCl₂. The transfection mix was incubated at roomtemperature for 25 minutes before it was added to the cells. HEK 293cells (ATCC CRL 1573) were grown in 2 cm by 2 cm coverglass chambers(Nunc, Denmark) with approximately 1.5 ml medium (Dulbecco's MEM withglutamax-1, 4500 mg/L glucose, and 10% FCS; Gibco BRL, Md., USA). TheDNA was added to cells at 25-50% confluence. Cells were grown at 37° C.in a CO₂ incubator. Prior to visualisation the medium was removed and1.5 ml Ca²⁺-HEPES buffer (5 mM KCl, 140 mM NaCl, 5.5 mM glucose, 1 mMMgSO₄, 1 mM CaCl, 10 mM HEPES) was added to the chamber.

Transfectants were visualised using an Axiovert 135 (Carl Zeiss,Germany) fluorescence microscope. The microscope was equipped with anHBO 100 mercury excitation source and a 40×, Fluar, NA=1.3 objective(Carl Zeiss, Germany). To visualise GFP, F64L-GFP, and F64L-S65T-GFP thefollowing filters were used: excitation 480/40 nm, dichroic 505 nm, andemission 510LP nm (all from Chroma Technologies Corp., Vt., USA). Tovisualise F64L-Y66H-GFP the following filters were used: excitation380/15 nm, dichroic 400 am, and emission 450/65 nm (all from OmegaOptical, Vt., USA).

Cells in several chambers were transfected in parallel, so that, a newchamber could be taken for each sample point. In cases where theincubation extended beyond 8.5 hours the Ca²⁺ precipitate was removed byreplacing the medium.

As shown in Table 1 the F64L mutation enhances the fluorescent signalsignificantly (wild type GFP versus F64L-GFP and F64L-S65T-GFP).Fluorescent cells can be observed as early as 1-2 hourspost-transfection indicating an efficient maturation of the chromophoreat 37° C. Furthermore, the F64L mutation is enhancing other GFPderivatives like the S65T mutant which has a shifted excitation spectrumand the blue derivative which is not detectable in mammalian cellswithout the F64L substitution. (Comment: When comparing the results ofF64L-S65T-GFP and F64L-GFP one has to take into account that theexcitation spectra differ and that the filter set used is optimised forF64L-S65T-GFP. F64L-GFP and WT GFP share the same spectral properties.)

23 1 36 DNA Aequorea victoria 1 tggaataagc tttatgagta aaggagaaga actttt36 2 36 DNA Aequorea victoria 2 aagaattcgg atccctttag tgtcaattgg aagtct36 3 67 DNA Aequorea victoria 3 ctacctgttc catggccaac gcttgtcactactttcctca tggtgttcaa tgcttttcta 60 gataccc 67 4 36 DNA Aequoreavictoria 4 aagaattcgg atccctttag tgtcaattgg aagtct 36 5 30 DNA Aequoreavictoria 5 aattggtacc aaggaggtaa gctttatgag 30 6 30 DNA Aequoreavictoria 6 ctttcgtttt gaattcggat ccctttagtg 30 7 48 DNA Aequoreavictoria 7 catggccaac gcttgtcact actctctctt atggtgttca atgctttt 48 8 48DNA Aequorea victoria 8 ctagaaaagc attgaacacc ataagagaga gtagtgacaagcgttggc 48 9 48 DNA Aequorea victoria 9 catggccaac gcttgtcactactctcactt atggtgttca atgctttt 48 10 48 DNA Aequorea victoria 10ctagaaaagc attgaacacc ataagtgaga gtagtgacaa gcgttggc 48 11 36 DNAAequorea victoria 11 tggaataagc tttatgagta aaggagaaga actttt 36 12 35DNA Aequorea victoria 12 gaatcgtaga tctttatttg tatagttcat ccatg 35 13 40DNA Aequorea victoria 13 tggaataagc tttatggatc ccgtcgtttt acaacgtcgt 4014 34 DNA Aequorea victoria 14 gcgcgaattc ttattattat ttttgacacc agac 3415 764 DNA Aequorea victoria CDS (8)..(721) 15 aagcttt atg agt aaa ggagaa gaa ctt ttc act gga gtt gtc cca att 49 Met Ser Lys Gly Glu Glu LeuPhe Thr Gly Val Val Pro Ile 1 5 10 ctt gtt gaa tta gat ggc gat gtt aatggg caa aaa ttc tcc gtt agt 97 Leu Val Glu Leu Asp Gly Asp Val Asn GlyGln Lys Phe Ser Val Ser 15 20 25 30 gga gag ggt gaa ggt gat gca aca tacgga aaa ctt acc ctt aaa ttt 145 Gly Glu Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe 35 40 45 att tgc act act ggg aag cta cct gtt ccatgg cca acg ctt gtc act 193 Ile Cys Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu Val Thr 50 55 60 act ctc tct cat ggt gtt caa tgc ttt tct agatac cca gat cat atg 241 Thr Leu Ser His Gly Val Gln Cys Phe Ser Arg TyrPro Asp His Met 65 70 75 aaa cag cat gac ttt ttc aag agt gcc atg ccc gaaggt tat gta cag 289 Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu GlyTyr Val Gln 80 85 90 gaa aga act ata ttt tac aaa gat gac ggg aac tac aagaca cgt gct 337 Glu Arg Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys ThrArg Ala 95 100 105 110 gaa gtc aag ttt gaa ggt gat acc ctt gtt aat agaatc gag tta aaa 385 Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg IleGlu Leu Lys 115 120 125 ggt att gat ttt aaa gaa gat gga aac att ctt ggacac aaa atg gaa 433 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Met Glu 130 135 140 tac aat tat aac tca cat aat gta tac atc atg gcagac aaa cca aag 481 Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala AspLys Pro Lys 145 150 155 aat ggc atc aaa gtt aac ttc aaa att aga cac aacatt aaa gat gga 529 Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn IleLys Asp Gly 160 165 170 agc gtt caa tta gca gac cat tat caa caa aat actcca att ggc gat 577 Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr ProIle Gly Asp 175 180 185 190 ggc cct gtc ctt tta cca gac aac cat tac ctgtcc acg caa tct gcc 625 Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu SerThr Gln Ser Ala 195 200 205 ctt tcc aaa gat ccc aac gaa aag aga gat cacatg atc ctt ctt gag 673 Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His MetIle Leu Leu Glu 210 215 220 ttt gta aca gct gct ggg att aca cat ggc atggat gaa cta tac aaa 721 Phe Val Thr Ala Ala Gly Ile Thr His Gly Met AspGlu Leu Tyr Lys 225 230 235 taaatgtcca gacttccaat tgacactaaa gggatccgaattc 764 16 238 PRT Aequorea victoria 16 Met Ser Lys Gly Glu Glu Leu PheThr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val AsnGly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr TyrGly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Ser His Gly Val Gln Cys PheSer Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys SerAla Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Tyr Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys GluAsp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 Tyr Asn SerHis Asn Val Tyr Ile Met Ala Asp Lys Pro Lys Asn Gly 145 150 155 160 IleLys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225230 235 17 764 DNA Aequorea victoria CDS (8)..(721) 17 aagcttt atg agtaaa gga gaa gaa ctt ttc act gga gtt gtc cca att 49 Met Ser Lys Gly GluGlu Leu Phe Thr Gly Val Val Pro Ile 1 5 10 ctt gtt gaa tta gat ggc gatgtt aat ggg caa aaa ttc tct gtt agt 97 Leu Val Glu Leu Asp Gly Asp ValAsn Gly Gln Lys Phe Ser Val Ser 15 20 25 30 gga gag ggt gaa ggt gat gcaaca tac gga aaa ctt acc ctt aaa ttt 145 Gly Glu Gly Glu Gly Asp Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe 35 40 45 att tgc act act ggg aag cta cctgtt cca tgg cca acg ctt gtc act 193 Ile Cys Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr 50 55 60 act ctc tct tat ggt gtt caa tgc ttttct aga tac cca gat cat atg 241 Thr Leu Ser Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met 65 70 75 aaa cag cat gac ttt ttc aag agt gcc atgccc gaa ggt tat gta cag 289 Lys Gln His Asp Phe Phe Lys Ser Ala Met ProGlu Gly Tyr Val Gln 80 85 90 gaa aga act ata ttt tac aaa gat gac ggg aactac aag aca cgt gct 337 Glu Arg Thr Ile Phe Tyr Lys Asp Asp Gly Asn TyrLys Thr Arg Ala 95 100 105 110 gaa gtc aag ttt gaa ggt gat acc ctt gttaat aga atc gag tta aaa 385 Glu Val Lys Phe Glu Gly Asp Thr Leu Val AsnArg Ile Glu Leu Lys 115 120 125 ggt att gat ttt aaa gaa gat gga aac attctt gga cac aaa atg gaa 433 Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile LeuGly His Lys Met Glu 130 135 140 tac aat tat aac tca cat aat gta tac atcatg gca gac aaa cca aag 481 Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile MetAla Asp Lys Pro Lys 145 150 155 aat ggc atc aaa gtt aac ttc aaa att agacac aac att aaa gat gga 529 Asn Gly Ile Lys Val Asn Phe Lys Ile Arg HisAsn Ile Lys Asp Gly 160 165 170 agc gtt caa tta gca gac cat tat caa caaaat act cca att ggc gat 577 Ser Val Gln Leu Ala Asp His Tyr Gln Gln AsnThr Pro Ile Gly Asp 175 180 185 190 ggc cct gtc ctt tta cca gac aac cattac ctg tcc acg caa tct gcc 625 Gly Pro Val Leu Leu Pro Asp Asn His TyrLeu Ser Thr Gln Ser Ala 195 200 205 ctt tcc aaa gat ccc aac gaa aag agagat cac atg atc ctt ctt gag 673 Leu Ser Lys Asp Pro Asn Glu Lys Arg AspHis Met Ile Leu Leu Glu 210 215 220 ttt gta aca gct gct ggg att aca catggc atg gat gaa cta tac aaa 721 Phe Val Thr Ala Ala Gly Ile Thr His GlyMet Asp Glu Leu Tyr Lys 225 230 235 taaatgtcca gacttccaat tgacactaaagggatccgaa ttc 764 18 238 PRT Aequorea victoria 18 Met Ser Lys Gly GluGlu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp GlyAsp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly AspAla Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly LysLeu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Ser Tyr Gly ValGln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp PhePhe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile PheTyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys PheGlu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 AspPhe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Pro Lys Asn Gly 145 150155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp GlyPro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser AlaLeu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu LeuGlu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu LeuTyr Lys 225 230 235 19 764 DNA Aequorea victoria CDS (8)..(721) 19aagcttt atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att 49 MetSer Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 1 5 10 ctt gtt gaatta gat ggc gat gtt aat ggg caa aaa ttc tct gtt agt 97 Leu Val Glu LeuAsp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser 15 20 25 30 gga gag ggtgaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt 145 Gly Glu Gly GluGly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 35 40 45 att tgc act actggg aag cta cct gtt cca tgg cca acg ctt gtc act 193 Ile Cys Thr Thr GlyLys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 50 55 60 act ctc act tat ggtgtt caa tgc ttt tct aga tac cca gat cat atg 241 Thr Leu Thr Tyr Gly ValGln Cys Phe Ser Arg Tyr Pro Asp His Met 65 70 75 aaa cag cat gac ttt ttcaag agt gcc atg ccc gaa ggt tat gta cag 289 Lys Gln His Asp Phe Phe LysSer Ala Met Pro Glu Gly Tyr Val Gln 80 85 90 gaa aga act ata ttt tac aaagat gac ggg aac tac aag aca cgt gct 337 Glu Arg Thr Ile Phe Tyr Lys AspAsp Gly Asn Tyr Lys Thr Arg Ala 95 100 105 110 gaa gtc aag ttt gaa ggtgat acc ctt gtt aat aga atc gag tta aaa 385 Glu Val Lys Phe Glu Gly AspThr Leu Val Asn Arg Ile Glu Leu Lys 115 120 125 ggt att gat ttt aaa gaagat gga aac att ctt gga cac aaa atg gaa 433 Gly Ile Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Met Glu 130 135 140 tac aat tat aac tca cataat gta tac atc atg gca gac aaa cca aag 481 Tyr Asn Tyr Asn Ser His AsnVal Tyr Ile Met Ala Asp Lys Pro Lys 145 150 155 aat ggc atc aaa gtt aacttc aaa att aga cac aac att aaa gat gga 529 Asn Gly Ile Lys Val Asn PheLys Ile Arg His Asn Ile Lys Asp Gly 160 165 170 agc gtt caa tta gca gaccat tat caa caa aat act cca att ggc gat 577 Ser Val Gln Leu Ala Asp HisTyr Gln Gln Asn Thr Pro Ile Gly Asp 175 180 185 190 ggc cct gtc ctt ttacca gac aac cat tac ctg tcc acg caa tct gcc 625 Gly Pro Val Leu Leu ProAsp Asn His Tyr Leu Ser Thr Gln Ser Ala 195 200 205 ctt tcc aaa gat cccaac gaa aag aga gat cac atg atc ctt ctt gag 673 Leu Ser Lys Asp Pro AsnGlu Lys Arg Asp His Met Ile Leu Leu Glu 210 215 220 ttt gta aca gct gctggg att aca cat ggc atg gat gaa cta tac aaa 721 Phe Val Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 taaatgtccagacttccaat tgacactaaa gggatccgaa ttc 764 20 238 PRT Aequorea victoria 20Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 1015 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 2530 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 4045 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 5560 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 7075 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 8590 95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys GlyIle 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met GluTyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys ProLys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn IleLys Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn ThrPro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr LeuSer Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg AspHis Met Ile Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr HisGly Met Asp Glu Leu Tyr Lys 225 230 235 21 764 DNA Aequorea victoria CDS(8)..(721) 21 aagcttt atg agt aaa gga gaa gaa ctt ttc act gga gtt gtccca att 49 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile 1 510 ctt gtt gaa tta gat ggc gat gtt aat ggg caa aaa ttc tct gtt agt 97Leu Val Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser 15 20 2530 gga gag ggt gaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt 145Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe 35 40 45att tgc act act ggg aag cta cct gtt cca tgg cca acg ctt gtc act 193 IleCys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr 50 55 60 actttc tct tat ggt gtt caa tgc ttt tca aga tac cca gat cat atg 241 Thr PheSer Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met 65 70 75 aaa cagcat gac ttt ttc aag agt gcc atg ccc gaa ggt tat gta cag 289 Lys Gln HisAsp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln 80 85 90 gaa aga actata ttt tac aaa gat gac ggg aac tac aag aca cgt gct 337 Glu Arg Thr IlePhe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala 95 100 105 110 gaa gtcaag ttt gaa ggt gat acc ctt gtt aat aga atc gag tta aaa 385 Glu Val LysPhe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys 115 120 125 ggt attgat ttt aaa gaa gat gga aac att ctt gga cac aaa atg gaa 433 Gly Ile AspPhe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu 130 135 140 tac aactat aac tca cat aat gta tac atc atg gca gac aaa cca aag 481 Tyr Asn TyrAsn Ser His Asn Val Tyr Ile Met Ala Asp Lys Pro Lys 145 150 155 aat ggaatc aaa gtt aac ttc aaa att aga cac aac att aaa gat gga 529 Asn Gly IleLys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly 160 165 170 agc gttcaa tta gca gac cat tat caa caa aat act cca att ggc gat 577 Ser Val GlnLeu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp 175 180 185 190 ggccct gtc ctt tta cca gac aac cat tac ctg tcc acg caa tct gcc 625 Gly ProVal Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala 195 200 205 ctttcc aaa gat ccc aac gaa aag aga gat cac atg atc ctt ctt gag 673 Leu SerLys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu 210 215 220 tttgta aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaa 721 Phe ValThr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235taaatgtcca gacttccaat tgacactaaa gggatccgaa ttc 764 22 238 PRT Aequoreavictoria 22 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val SerGly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His MetLys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr ArgAla Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Met Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met AlaAsp Lys Pro Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Lys Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn GluLys Arg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 23 14 DNA ArtificialSequence DNA sequence at the lacZ-promoter GFP fusion point 23aggaaagctt tatg 14

What is claimed is:
 1. A nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) polypeptide that has the amino acid sequence of SEQ ID NO:22 with the exception that a Leu residue is substituted for the Phe residue at position 64 of SEQ ID NO:22.
 2. A nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) polypeptide that has the amino acid sequence of SEQ ID NO:22 with the exception that an amino acid residue selected from the group consisting of Leu, Ile, Val, Gly and Ala is substituted for the Phe residue at position 64 of SEQ ID NO:22.
 3. The nucleic acid molecule according to claim 2 wherein a Leu residue is substituted for the Phe residue at position 64 of SEQ ID NO:22 which is further substituted in that a His residue is substituted for the Tyr residue at position 66 of SEQ ID NO:22.
 4. The nucleic acid molecule according to claim 2 wherein a Ile residue is substituted for the Phe residue at position 64 of SEQ ID NO:22 which is further substituted in that a His residue is substituted for the Tyr residue at position 66 of SEQ ID NO:22.
 5. The nucleic acid molecule according to claim 2 wherein a Ala residue is substituted for the Phe residue at position 64 of SEQ ID NO:22 which is further substituted in that a His residue is substituted for the Tyr residue at position 66 of SEQ ID NO:22.
 6. The nucleic acid molecule according to claim 2 wherein a Val residue is substituted for the Phe residue at position 64 of SEQ ID NO:22 which is further substituted in that a His residue is substituted for the Tyr residue at position 66 of SEQ ID NO:22.
 7. The nucleic acid molecule according to claim 2 wherein a Gly residue is substituted for the Phe residue at position 64 of SEQ ID NO:22 which is further substituted in that a His residue is substituted for the Tyr residue at position 66 of SEQ ID NO:22.
 8. A nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) having the amino acid sequence of SEQ ID NO:
 16. 9. A nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) having the amino acid sequence of SEQ ID NO:
 18. 10. A nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) having the amino acid sequence of SEQ ID NO:
 20. 11. An expression vector comprising suitable expression control sequences operatively linked to a nucleic acid molecule according to claim
 1. 12. A recombinant host cell comprising an expression vector that comprises suitable expression control sequence operatively linked to a nucleic acid molecule according to claim
 1. 13. A nucleic acid molecule comprising a nucleotide sequence encoding a protein of interest, wherein said nucleic acid is fused to the nucleotide sequence encoding a Green Fluorescent Protein (GFP) according to claim
 1. 14. A nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) having an amino acid sequence in which the amino acid Phe immediately upstream of the chromophore is substituted with an amino acid selected from the group consisting of Leu, Ile, Val, Gly, and Ala, wherein said chromophore has an amino acid sequence selected from the group consisting of SerTyrGly, SerHisGly, ThrHisGly and ThrTyrGly, and wherein said substituted GFP exhibits increased fluorescence at the same wavelength at a temperature of 30° C. or above, relative to a GFP lacking the above substitution, when expressed in a host cell.
 15. An expression vector comprising suitable expression control sequences operatively linked to a nucleic acid molecule according to claim
 14. 16. A recombinant host cell comprising an expression vector that comprises suitable expression control sequence operatively linked to a nucleic acid molecule according to claim
 14. 17. A nucleic acid molecule comprising a nucleotide sequence encoding a protein of interest, wherein said nucleic acid is fused to the nucleotide sequence encoding a Green Fluorescent Protein (GFP) according to claim
 14. 18. A nucleic acid molecule according to claim 14, wherein a Leu residue is substituted for the Phe residue at position 64 of SEQ ID NO:
 22. 19. The nucleic acid molecule according to claim 14, wherein a Leu residue is substituted for the Phe residue.
 20. The nucleic acid molecule according to claim 14, wherein a Ile residue is substituted for the Phe residue.
 21. The nucleic acid molecule according to claim 14, wherein a Ala residue is substituted for the Phe residue.
 22. The nucleic acid molecule according to claim 14, wherein a Val residue is substituted for the Phe residue.
 23. The nucleic acid molecule according to claim 14, wherein a Gly residue is substituted for the Phe residue.
 24. The nucleic acid molecule according to claim 14, wherein said substituted GFP exhibits increased fluorescence at the same wavelength at a temperature of from 32° C. to 39° C.
 25. The nucleic acid molecule according to claim 14, wherein said substituted GFP exhibits increased fluorescence at the same wavelength at a temperature of from 35° C. to 38° C.
 26. The nucleic acid molecule according to claim 14, wherein said substituted GFP exhibits increased fluorescence at the same wavelength at a temperature of about 37° C.
 27. The nucleic acid molecule according to claim 14, wherein said GFP is derived from Aequoria victorea or Renilla reniformis.
 28. The nucleic acid molecule according to claim 14, wherein said unsubstituted GFP is obtained from Aequoria victorea.
 29. The nucleic acid molecule according to claim 14, wherein said unsubstituted GFP is obtained from Renilla reniformis.
 30. The nucleic acid molecule according to claim 14, wherein said GFP has the amino acid sequence of SEQ ID NOs: 16, 18, 20 or
 22. 31. A nucleic acid molecule comprising a nucleotide sequence encoding a Green Fluorescent Protein (GFP) comprising an amino acid sequence in which the amino acid immediately upstream of the chromophore is substituted with an amino acid selected from the group consisting of Leu, Ile, Val, Gly, and Ala, wherein said chromophore has an amino acid sequence selected from the group consisting of SerTyrGly, SerHisGly, ThrHisGly and ThrTyrGly, and wherein said substituted GFP exhibits increased fluorescence at the same wavelength at a temperature of 30° C. or above, relative to a GFP lacking the above substitution, when expressed in a host cell. 